The bioavailability of drugs is a complex issue. For a long time efforts were focussed on the processes occurring in the liver when addressing the issue of bioavailability of drugs. All blood from the gastrointestinal tract passes the liver before going anywhere else in the body. Thus, first pass effect of the liver was thought to be of great influence on bioavailability. Certainly it was thought to be of more influence than any mechanism exerted by the gut. This was thought to be the case for example due to the much lower presence of cytochrome P450 in the gut as compared to the liver. It was known for a fact that cytochrome P450 catalyses phase I biotransfornation, i.e. the process involved in removal of drugs from the body. In phase II, the subsequent step of the removal process, a hydrophilic group is added in order to increase solubility and thus subsequently speed up elimination through bile or kidneys.
Traditionally, efforts have thus been focussed on increasing solubility and membrane permeability when addressing the problem of bioavailability of drugs. Most particularly, metabolism-associated approaches have been focussed on the liver biotransformation process. The problem with these approaches has, however, been the broad effects on liver metabolism in general and thus broad non-specific and often thus undesirable systemic effects.
Recently however, it has been suggested that the absorption across intestinal epithelia also affect bioavailability of drugs. The enterocyte membrane contains numerous transport proteins that carry nutrients from the lumen of the gut into the enterocytes. Active or passive transport through the membrane is responsible for the passage of many molecules through the membrane into the cytoplasm. The nutrients, and also drugs, subsequently pass through the enterocytes into the capillary net and proceed to the circulation system and the liver.
However, the intestine can also remove compounds from the cytoplasm of enterocytes and transport these compounds back to the lumen. Presumably this is a mechanism that has evolved to protect against potentially damaging compounds that enter the body via the oral route. Following this line of reasoning, U.S. Pat. No. 5,567,592 (equivalent to WO 95/20980) suggests that two gut related mechanisms could be inhibited in order to increase the net flux of drug from the gut. On the one hand an inhibitor of cytochrome P450, in particular inhibitors of the cytochrome P 450 3A (CYP3A) is suggested and on the other hand use of an inhibitor of P-glycoprotein (P-gp) or a combination of the two categories of inhibitors is suggested. It is worth noting that inhibitors of P-gp and CYP3A are extensively disclosed in the prior art. Moreover, inhibitors of CYP3A are generally hydrophobic compounds that can pass cell membranes without the need of transport proteins.
P-glycoprotein back transport activity in the gut of a mammal can be inhibited with a view to increasing drug bioavailability by virtue of the fact that the net transport of drugs through the enterocyte layer will be enhanced. P-glycoprotein is located inter alia in the small intestine and colon on the luminal side of epithelial cells and transports dietary toxins back into the lumen and thus helps prevent the toxins being absorbed into the portal circulation.
U.S. Pat. No. 5,567,592 does, however, not illustrate or give a specific example of a P-gp inhibitor. Nor does it provide any information on success of the chosen method. The document merely shows an increase in cyclosporin bioavailability caused by co-administration of ketoconazole. Ketoconazole is a cytochrome P450 3A inhibitor.
A later document (The Lancet, Vol. 352, Jul. 25, 1998) describes how coadministration of cyclosporin enables oral therapy with paclitaxel in patients. Normally, orally introduced paclitaxel is poorly bioavailable due to the exceedingly high affinity thereof for the multidrug transporter P-glycoprotein which is abundantly present in the gastrointestinal tract (Trends Genet. 1997; 13:217–22, Cell 1994; 77: 491–502). Studies in mdr1a (−/−) knock out mice lacking P-gp revealed an increased uptake of paclitaxel (Proc. Natl. Acad. Sci. U.S.A. 1997; 4:2031–35) Subsequently, it was thus described (British Journal of Cancer 1997; 76: 1181–1183) in how paclitaxel was orally introduced into wild type mice together with the P-gp blocker SDZ PSC 833 or the P-gp blocker cyclosporin (Clinical Cancer Research 1998; 4: 2293–2297) resulting in a tenfold increased systemic exposure to paclitaxel. Proof of concept tests were subsequently carried out on patients (The Lancet, Vol. 352, Jul. 25, 1998) and confirmed the results. Co-administration of paclitaxel with cyclosporin increased the absorption of oral paclitaxel to therapeutic plasma concentrations.
The P-gp is known from its association with multi drug resistance development of tumor cells. A number of other transport proteins have also been associated with multi drug resistance (MDR) such as the MRP (multidrug resistance associated protein) and possibly the MVP (major vault protein). An alternative system also leading to MDR is the interference of some drugs in the ability of the cell to enter apoptosis for example cells genetically deficient in p53 or cells overexpressing bclxL. Both MRP and P-gp belong to the group of proteins classified as ABC proteins. ABC proteins function by way of their being ATP binding proteins. The phenomenon of multi drug resistance consists of tumor cells exhibiting resistance to a large number of structurally unrelated antineoplastic agents. These agents include anthracyclines, vinca alkaloids, taxol and epipodophyllotoxins. ATP hydrolysis on the cytoplasmic face of P-gp is required for transport of hydrophobic compounds from a tumor cell. The addition of verapamil, diltiazem, quinine, trifluoperazine or cyclosporin seems to potentially reverse P-gp asssociated MDR.
It should be noted that in spite of extensive knowledge of these MDR mechanisms in iii vitro systems in many tumors, it is still unclear what mechanisms contribute most to the multi drug resistance in the clinical setting. It is quite possible that other unidentified or poorly understood MDR mechanisms will turn out to be at least as important as the MDR mechanisms defined above.
In this respect we point to a new protein that has been found. The protein is called Breast Cancer Resistance Protein or BCRP. It is also known as MXR or ABCP. A number of recent publications have illustrated that this protein is also a drug resistance related protein. A number of such disclosures are provided in the Proceedings of the American Association for Cancer Research volume 40, March 1999. e.g. Rocchi et al. Abstract 2090, Zhan et al. Abstract 2091, Ross et al. Abstract 2092, Rabindran et al. Abstract 2093, Litman et al. Abstract 4413, Schlegel et al. Abstract 4415, Rohde et al. Abstract 4417 and Rabindran et al., Cancer Research 1998; 58: 5850–5858.
The amino acid sequence of BCRP has been determined and the gene has been isolated and sequenced (Doyle et al. Proc. Natl. Acad. Sci USA 1998; Vol 95; 15665–15670 and WO 99/40110). It has been determined to be an ABC transport protein. The P-gp protein is also an ABC protein, but differs significantly from the BCRP. This is clearly illustrated by the sequence data and also by the fact that the presence of verapamil (an inhibitor of P-gp) did not prevent drug resistance for doxorubicin in cells overexpressing BCRP. The doxorubicin resistance was subsequently attributed to the overexpression of this protein. Thus cells exhibiting doxorubicin resistance can possess either P-gp and/or BCRP transport mechanisms. Also on the other hand P-gp overexpressing cells exhibit resistance to paclitaxel and vincristine. No resistance to these compounds is however present when the P-gp mechanism is inactive and the BCRP mechanism is active. Thus there are clearly two different systems of drug resistance with different proteins that show different specificities to drugs.
Rabindran et al. (Proc. Am. Assoc. Cancer Res.; 40: abstract 2093 and Cancer. res. 1998; 58: 5850–5858) disclose that the mycotoxin fumitremorgin C (FTC) reverses in vitro non-P-gp, non-MRP-mediated MDR in mitoxantrone-selected cells derived from a human colon carcinoma cell line. It was found that FTC did not reverse MDR in cells overexpressing P-gp or MRP. It was therefore suggested that this reversal of non-P-gp, non-MRP-mediated MDR involved a transport protein, possibly BCRP, having substrate specificities substantially different from those of P-gp and MRP. Such a suggestion can merely be considered speculative in view of the complexity of the issues as is illustrated on page 5857 of this article, second paragraph where it is stated that “the mechanism by which FTC reverses drug resistance is unknown”. This article is further silent regarding any link between BCRP and non-tumor cells. In addition, in vivo data are not provided.
Hazlehurst et al. (Cancer. Res. 1999; 59: 1021–1027) disclose that at low levels of MDR FTC reverses in vitro MDR in mitoxantrone-selected cells derived from the P-pg negative human myeloma cell line 8226. This reversal was attributed to BCRP. However, at higher levels of MDR several other drug-resistant mechanisms can be involved including non-transport phenomena as evidenced by reduced topoisomerase II levels and activity. It remains therefore uncertain whether the reversal can in fact be attributed specifically to BCRP and certainly is uncertain in the case of higher levels of MDR then were tested. Regarding this issue it is worth noting that increasing the degree of resistance to mitoxantrone in the human 8226/MR20 myeloma cell line from 10 to 37 times did not further reduce the intracellular drug concentration. Additionally, there is no suggestion or teaching of BCRP in relation to non-tumor cells. Furthermore, in vivo data are not provided.
Consequently, the prior art only relates to MDR in tumor cells and is silent about drug transport in normal cells. Additionally, much speculation exists about the mechanisms involved in MDR. Furthermore, the studies are limited to in vitro systems. Forms of administration of drugs, in particular oral administration of drugs, in relation to drug transport in normal cells, is not addressed.
WO 99/40110 (priority date 5 Feb. 1998, published 12 Aug. 1999; WO 99/40110 is a non-prepublished patent application and therefore only relevant for novelty) discloses BCRP that is overexpressed in breast carcinoma cell lines, inhibitors of BCRP such as immunoglobulins (e.g. antibodies) and non-immuno-globulins (e.g. organic compounds such as FTC). Example 14 shows the beneficial effect of FTC on the intercellular concentration of BBR 3390 in MCF-7 cells (a human breast carcinoma cell line). There is no disclosure of the oral administration of BCRP inhibitors together with pharmaceutical compounds to enhance the bioavailability of the latter. There is no link mentioned between normal tissue, i.e. healthy tissue, and BCRP. Additionally, only in vitro data are provided for the effect of FTC on the inter- and intracellular BBR 3390 concentrations in MCF-7 cells.
Notwithstanding the lack of data concerning the BCRP and the mechanism of transport, it was decided to investigate whether it could be a useful target to approach with a view to increasing oral bioavailability of drugs. An analogous line of reasoning to that employed for P-gp was followed, however, without any detailed knowledge of the mechanism to be inhibited and the potential consequences thereof for the cell or more importantly the patient. Furthermore, there was no knowledge on whether the effect would be high enough to show any effect on the drug distribution. Neither was there any knowledge whether inhibition of such a transport system would result in activation of another system. Nor was there any indication whether the inhibition would be more harmful than beneficial. Due to the large differences between P-gp and BCRP and lack of knowledge concerning the transport mechanism, there was no reasonable expectation of success that the BCRP system might function analogously to that of P-gp and thus that inhibition thereof could increase oral drug delivery without potentially seriously disrupting the normal cellular processes and thus potentially being detrimental to the patient.